2',7'-Dichlorofluorescein NHS (DCF)

2',7'-Dichlorofluorescein (DCF) NHS ester is an amine-reactive fluorescent dye widely used for the post-synthetic labeling of amino-modified oligonucleotides. The N-hydroxysuccinimide (NHS) ester selectively reacts with primary amines introduced at the 5' end, 3' end, or internal positions of DNA, RNA, PNA, or other oligonucleotide analogs, producing a stable amide bond. This straightforward conjugation chemistry allows high labeling efficiency while preserving the integrity of the oligonucleotide.

DCF is a chlorinated derivative of fluorescein that exhibits bright green fluorescence with excitation and emission maxima of approximately 503 nm and 528 nm, respectively. Compared with fluorescein, the dichloro substitution slightly alters the spectral properties and can improve photophysical performance in certain assay conditions. The dye is readily detected using standard FITC filter sets found on fluorescence microscopes, plate readers, flow cytometers, and many molecular imaging instruments.

DCF-labeled oligonucleotides are commonly used in:

• Fluorescence in situ hybridization (FISH)
• RNA and DNA imaging
• Hybridization assays
• Real-time and endpoint fluorescence detection
• Flow cytometry
• Cellular uptake studies
• Microarray applications
• Biosensor development
• Fluorescent affinity assays

For optimal labeling efficiency, the oligonucleotide should contain a primary amino group, typically introduced through 5'-Amino Modifier, 3'-Amino Modifier, or an internal amino linker such as Amino C6 dT. NHS ester conjugation is generally performed in mildly basic buffers (typically pH 8.3–9.0) that promote efficient reaction with primary amines while minimizing hydrolysis of the NHS ester. Following conjugation, excess free dye is removed by HPLC, desalting, or other purification methods to produce highly purified fluorescent oligonucleotides.

Bio-Synthesis offers DCF NHS labeling on a wide range of custom DNA, RNA, PNA, LNA, siRNA, antisense oligonucleotides, PCR probes, imaging probes, and other modified oligonucleotides. DCF can be combined with numerous additional modifications, including quenchers, biotin, digoxigenin, click chemistry handles, peptides, GalNAc, lipids, and other conjugates, enabling multifunctional probe and therapeutic research designs.

Typical Properties

Recommended Amino Modifiers

• 5'-Amino Modifier C6
• 5'-Amino Modifier C12
• 3'-Amino Modifier C3
• 3'-Amino Modifier Serinol
• Internal Amino C6 dT
• Internal Amino C12 dT

Product Information

 

Product Name:

2',7'-Dichlorofluorescein NHS (DCF)

Alternate Name:

2',7'-Dichlorofluorescein NHS

Category:

Fluorescent Dye

Modification Code:

[5-DCF], [3-DCF],[iDCF-dT]

Excitation (nm):

503

Emission (nm):

528

Structure:

Purification:

dual HPLC

Delivery Format:

Lyophilized

Shipping Conditions:

Room Temperature

Storage Conditions:

-20°C To -70°C
Oligonucleotides are stable in solution at 4°C for up to 2 weeks. Properly reconstituted material stored at -20°C should be stable for at least 6 months. Dried DNA (when kept at 20°C) in a nuclease-free environment should be stable for years.

References/Citations:

Selected References

1. Hermanson GT. Bioconjugate Techniques. 3rd ed. Academic Press; 2013. View reference
2. Haugland RP. The Handbook—A Guide to Fluorescent Probes and Labeling Technologies. Invitrogen / Molecular Probes.
3. Brinkley M. A brief survey of methods for preparing protein conjugates with dyes, haptens, and crosslinking reagents. Bioconjugate Chemistry. 1992;3(1):2-13. DOI: 10.1021/bc00013a001
4. Thermo Fisher Scientific. Amine-Reactive NHS Ester Crosslinker Chemistry. Technical resource on NHS ester reaction with primary amines. View resource

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